Optimization of dipeptide-linked benzimidazole topoisomerase 1 poisons

描述（由申请人提供）：喜树碱（CPT）的衍生物抑制拓扑异构酶I （Top1）的活性，已证明在治疗各种癌症的临床应用。然而，CPT药物特性的局限性促使人们寻找靶向Top1蛋白- dna切割复合物的新型CPT样毒药。我们假设，同时结合DNA小槽和Top1蛋白的鼻锥区域的双功能剂将是有效的毒药，具有优于CPT同系物的药物特性。这类化合物对那些对CPT结构类毒物具有抗性的肿瘤也应该有效，并且与拓扑替康等CPT类药剂相比，通常应该表现出更低的毒性。利用Top1-DNA晶体结构的结构见解设计了一个与Hoechst 33258（一种DNA小凹槽粘合剂）连接的n -乙酰基二肽小文库。对文库进行体外筛选和随后的文库反卷积得到了一种高亲和力（EC50 ~ 50 nM）的试剂LL217，该试剂也引起了几种肿瘤细胞系的生长停滞。随后用苯基取代的单苯并咪唑替代Hoechst，使MB1表现出与LL217相似的肿瘤细胞系杀伤活性，但脱靶效应大大降低。用LL217和MB1进行的生化分析表明，在肿瘤细胞系中的主要作用方式是通过抑制Top1活性来阻止细胞周期。我们的长期目标是将MB1转化为临床有效的药物。我们的近期目标是生产一种具有足够组织培养和小鼠异种移植数据的精制先导化合物，以支持进一步的开发。近期目标将通过四个具体目标来实现：(1)MB1的结构将被稍微修改以促进固相合成；(2)MB1衍生物的体外和细胞活性将被分析并与拓扑替康进行比较；(3)MB1的潜在治疗效果将在小鼠异种移植肿瘤模型中与拓扑替康进行比较；(4)MB1和拓扑替康的药物特性将被评估。公共卫生相关性：癌症最近已经超过心血管疾病，成为美国发病率和死亡率的主要原因。虽然需要新的化疗药物类别，但开发具有改进药物特性的药物以临床验证的靶点为目标也是可取的。临床上使用的喜树碱类药物专门针对拓扑异构酶1。然而，它们的溶解度和稳定性差，需要使用大剂量，这增加了对患者产生不良反应的机会。这里描述的药物也靶向拓扑异构酶1，但它们有望具有更高的生物利用度，因此，对喜树碱类药物进行了改进。由于这些药物在结构上也不同，因此预计它们在临床上对已经对喜树碱产生耐药性的癌症有效。

DESCRIPTION (provided by applicant): Derivatives of camptothecin (CPT) that inhibit Topoisomerase I (Top1) activity have demonstrated clinical utility in the treatment of various cancers. However, limitations in CPT pharmaceutical properties have driven the search for new CPT-like poisons that target the Top1 protein-DNA cleavage complex. We hypothesized that bifunctional agents that simultaneously bind to the DNA minor groove and the nose-cone region of the Top1 protein would be effective poisons with pharmaceutical properties that are superior to the CPT congeners. Such compounds also should be effective against tumors that are otherwise resistant to the CPT structural class of poisons and should generally exhibit reduced toxicity compared to the CPT- class of agents such as topotecan. Structural insights from Top1-DNA crystal structures were used to design a small library of N-acetyl dipeptides linked to Hoechst 33258, a DNA minor groove binder. Screening of the library against a in vitro assays and subsequent library deconvolution afforded a high affinity (EC50 ~ 50 nM) agent, LL217, that also caused growth arrest of several tumor cell lines. Subsequent replacement of the Hoechst with a phenyl- substituted monobenzimidazole provided the agent MB1 that exhibits tumor cell line killing activity similar to LL217 but with substantially reduced off-target effects. Biochemical assays performed with LL217 and MB1 demonstrate that the primary modality of action in tumor cell lines is cell cycle arrest due to inhibition of Top1 activity. Our long-term goal is to translate MB1 into a clinically efficacious drug. Our immediate goal is to produce a refined lead compound with sufficient tissue culture and murine xenograft data to support further development. The immediate goals will be achieved via four specific aims: (1) the structure of MB1 will be slightly modified to facilitate solution phase syntheses, (2) the in vitro and cellular activities of the MB1 derivatives will be assayed and compared to topotecan, (3) the potential therapeutic efficacy of MB1 will be evaluated in murine xenograft tumor models as compared to topotecan, and (4) the pharmaceutical properties of MB1 and topotecan will be evaluated. PUBLIC HEALTH RELEVANCE: Cancer has recently surpassed cardiovascular disease as a major cause of morbidity and mortality in the United States. Although new classes of chemotherapeutic agents are needed, it is also desirable to develop agents with improved pharmaceutical properties that target clinically validated targets. The camptothecin class of agents used in the clinic specifically target the Topoisomerase 1 enzyme. However, their poor solubilities and stabilities require the use of large doses, which increases the chances for adverse effects to the patient. The agents described here also target Topoisomerase 1, but they are expected to be more bioavailable and, thus, offer an improvement to the camptothecin class of agents. Because these agents are also structurally distinct, it is expected that they will be clinically efficacious against cancers that have developed resistance to the camptothecins.